Multiple domains are involved in the targeting of the mouse DNA methyltransferase to the DNA replication foci.
It has been shown that, during the S-phase of the cell cycle, the mouse DNA methyltransferase (DNA MTase) is targeted to sites of DNA replication by an amino acid sequence (aa 207-455) lying in the N-terminal domain of the enzyme [Leonhardt, H., Page, A. W., Weier, H. U. and Bestor, T. H. (1992) Cell , 71, 865-873]. In this paper it is shown, by using enhanced green fluorescent protein (EGFP) fusions, that other peptide sequences of DNA MTase are also involved in this targeting. The work focuses on a sequence, downstream of the reported targeting sequence (TS), which is homologous to the Polybromo-1 protein. This motif (designated as PBHD) is separated from the reported targeting sequence by a zinc-binding motif [Bestor , T. H. (1992) EMBO J , 11, 2611-2617]. Primed in situ extension using centromeric-specific primers was used to show that both the host DNA MTase and EGFP fusion proteins containing the targeting sequences were localized to centromeric, but not telomeric, regions during late S-phase and mitosis. Also found was that, in approximately 10% of the S-phase cells, the EGFP fusions did not co-localize with the centromeric regions. Mutants containing either, or both, of these targeting sequences could act as dominant negative mutants against the host DNA MTase. EGFP fusion proteins, containing the reported TS (aa 207-455), were targeted to centromeric regions throughout the mitotic stage which lead to the discovery of a similar behavior of the endogenous DNA MTase although the host MTase showed much less intense staining than in S-phase cells. The biological role of the centromeric localization of DNA MTase during mitosis is currently unknown.
Liu
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Y
Friedrich Miescher-Institut, P.O. Box 2543, Basel, Switzerland.
Oakeley
E J
EJ
Sun
L
L
Jost
J P
JP
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002503:Centromere / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D002843:Chromatin; D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015254:DNA Modification Methylases